In the production of nucleic acid substances such as inosine, guanosine and bases thereof (hypoxanthine and guanine and the like) by fermentation, there have conventionally been used mutant strains imparted with adenine auxotropy and nucleic acid analogue resistance (Japanese Patent Publication (KOKOKU) Nos. Sho 55-2956 and Sho 55-45199) with a limited amount of adenine substances in their culture medium.
Mutant strains obtained by usual mutagenesis procedures are often introduced with mutations in their genes other than the target gene. In addition, because complicated regulation mechanisms are involved in biosynthetic pathways of nucleic acid substances, it is difficult to obtain a microorganism that produces a significant amount of nucleic acid substance. Therefore, mutant strains obtained by conventional breeding methods have not necessarily been satisfactory ones.
On the other hand, there has been disclosed a method for producing inosine or guanosine by utilizing a bacterium belonging to the genus Bacillus exhibiting enhanced purine operon expression obtained by modification of a gene sequence encoding enzymes involved in the purine biosynthesis (purine operon) (Japanese Patent Unexamined Publication (KOKAI) No. Hei 3-164185). This method is characterized in that a promoter or operator of the purine operon is modified to increase the expression level of the operon, thereby increasing the production of inosine or guanosine.
Expression of the purine operon of Bacillus subtilis (purEKBC(ORF)OLFMNHD where ORF represents an open reading frame of unknown function) is suppressed by an excessive amount of adenine, and also regulated by attenuation caused by guanine. Further, a repressor protein binding to the 5' flanking region of the purine operon and a gene encoding the protein (purR) have been isolated, and it has been reported that, in a Bacillus subtilis strain whose purR gene was disrupted, suppression by adenine as for the expression of purC-lacZ fusion gene integrated into the purine operon was reduced to about 1/10 (Proc. Natl. Acad. Sci. USA, 92, 7455-7549 (1995)).
In Bacillus subtilis, the repressor protein has been known to regulate the expression of, in addition to the genes of the purine operon, purA gene involved in the biosynthesis of adenine and genes of pyrimidine operon involved in the pyrimidine biosynthesis (1997, J. Bacteriol. 179, 7394-7402, H. Zalkin).
On the other hand, in Escherichia coli, it has been reported that the purine operon repressor also affects the expression of glyA gene involved in the biosynthesis of glycine, which is a substance of 5'-IMP (inosinic acid) biosynthesis (1990, J. Bacteriol. 172, 3799-3803, H. Zalkin et al.), and the expression of gcv operon genes involved in the production of C.sub.1 and CO.sub.2 supplied from glycine (1993, J. Bacteriol. 175, 5129-5134, G. V. Stauffer et al.) in addition to the genes of the purine operon.
As described above, several reports have been made about the relationship between the purine operon and inosine or guanosine production. However, there are various biosynthesis pathways involving the purR gene. Further, the purine operon of Bacillus subtilis encodes ten enzymes, and involved in many reactions. Thus, the biosynthesis pathways of nucleic acid substances are very complicated, and the relationship between the purR gene and accumulation of nucleic acid substances has scarcely been known.